1. Field of the Invention
The present invention relates to a measuring apparatus, such as a surface plasmon resonance measuring apparatus, for analyzing a substance in a sample by utilizing the excitation of surface plasmon, and more particularly to a measuring apparatus for analyzing a substance in a sample, by making a light beam enter the interface between a thin film layer (or a metal film, or a cladding layer) in contact with the sample and a dielectric block so that the light beam is totally reflected at the interface, making an evanescent wave occur, and measuring a change in the intensity of the totally reflected light beam due to the evanescent wave. The invention also relates to a measuring chip that is employed in such a measuring apparatus.
2. Description of the Related Art
In metals, if free electrons are caused to vibrate in a group, a compression wave called a plasma wave will be generated. The compression wave, generated in the metal surface and quantized, is called surface plasmon.
There have hitherto been proposed various kinds of surface plasmon resonance measuring apparatuses for quantitatively analyzing a substance in a sample by taking advantage of a phenomenon that surface plasmon is excited by a light wave. Among such apparatuses, one employing a system called “Kretschmann configuration” is particularly well known (e.g., see Japanese Unexamined Patent Publication No. 6(1994)-167443).
The surface plasmon resonance measuring apparatus employing the “Kretschmann configuration” is equipped with a dielectric block formed, for example, into the shape of a prism; a metal film, formed on one surface of the dielectric block, for placing a sample thereon; and a light source for emitting a light beam. The measuring apparatus is further equipped with an optical system for making the light beam enter the dielectric block so that a condition for total internal reflection (TIR) is satisfied at the interface between the dielectric block and the metal film and that various angles of incidence, including a surface plasmon resonance condition, are obtained; and photodetection means for measuring the intensity of the light beam totally reflected at the interface, thereby detecting surface plasmon resonance.
To obtain various angles of incidence in the aforementioned manner, a relatively thin light beam can be deflected so that it strikes the above-mentioned interface at different angles of incidence, or a relatively thick beam can be convergently emitted so that the components thereof strike the interface at various angles of incidence. In the former, the light beam whose reflection angle varies with the deflection thereof can be detected by a small photodetector that is moved in synchronization with the light beam deflection, or by an area sensor extending along a direction where the reflection angle varies. In the latter, on the other hand, the light beams reflected at various angles can be detected by an area sensor extending in a direction where all the reflected light beams are received.
In the surface plasmon resonance measuring apparatus mentioned above, an evanescent wave having electric field distribution is generated in a sample in contact with the metal film, if a light beam strikes the metal film at a specific incidence angle θsp greater than a critical incidence angle at which total internal reflection (TIR) takes place. The generated evanescent wave excites surface plasmon at the interface between the metal film and the sample. When the wave vector of the evanescent wave is equal to the wave number of the surface plasmon and therefore the wave numbers between the two are matched, the evanescent wave resonates with the surface plasmon and the light energy is transferred to the surface plasmon, whereby the intensity of the light satisfying TIR at the interface between the dielectric block and the metal film drops sharply. This sharp intensity drop is generally detected as a dark line by the above-mentioned photodetection means.
Note that the above-mentioned resonance occurs only when an incident light beam is a p-polarized light beam. Therefore, in order to make the resonance occur, it is necessary that a light beam be p-polarized before it strikes the interface.
If the wave number of the surface plasmon is found from the specific incidence angle θsp at which attenuated total reflection (ATR) takes place, the dielectric constant of a sample to be analyzed can be calculated by the following Equation:Ksp(ω)=(ω/c){∈m(ω)∈s}1/2/{∈m(ω)+∈s}1/2where Ksp represents the wave number of the surface plasmon, ω represents the angular frequency of the surface plasmon, c represents the speed of light in vacuum, and ∈m and ∈s represent the dielectric constants of the metal and the sample, respectively.
If the dielectric constant ∈s of a sample is found, the density of a specific substance in the sample is found based on a predetermined calibration curve or the like. As a result, the specific substance in the sample can be quantitatively analyzed by finding the specific incidence angle θsp at which the intensity of the reflected light at the interface drops sharply.
In the conventional surface plasmon resonance measuring apparatus employing the aforementioned system, the metal film on which a sample is placed must be exchanged for a new one each time a measurement is made. Because of this, the metal film and the dielectric block are integrated into a single measuring unit (chip). After a measurement of each sample is made, each measuring unit is discarded (e.g., see Japanese patent application No. 2001-016633, filed by the present applicant)
As a similar sensor making use of ATR, there is known a leaky mode sensor (e.g., see “Spectral Researches,” Vol. 47, No. 1 (1998), pp. 21 to 23 and pp. 26 to 27). This leaky mode sensor is equipped with a dielectric block formed, for example, into the shape of a prism; a cladding layer formed on one surface of the dielectric block; and an optical waveguide layer, formed on the cladding layer, for placing a sample thereon. The leaky mode sensor is further equipped with a light source for emitting a light beam; an optical system for making the light beam enter the dielectric block at various angles of incidence so that a condition for total internal reflection (TIR) is satisfied at the interface between the dielectric block and the cladding layer and so that ATR occurs by a waveguide mode excited in the optical waveguide layer; and photodetection means for measuring the intensity of the light beam totally reflected at the interface between the dielectric block and the cladding layer, and detecting the excited state of the waveguide mode, that is, ATR.
In the leaky mode sensor mentioned above, if a light beam strikes the cladding layer through the dielectric block at incidence angles greater than a critical incidence angle at which TIR takes place, the light beam is transmitted through the cladding layer and then only light with a specific wave number, incident at a specific incidence angle, propagates through the optical waveguide layer in a waveguide mode. If the waveguide mode is excited in this manner, the greater part of the incident light is confined within the optical waveguide layer, and consequently, ATR occurs in which the intensity of light totally reflected at the above-mentioned interface drops sharply. Since the wave number of the light propagating through the optical waveguide layer depends on the refractive index of a sample on the optical waveguide layer, both the refractive index of the sample and the properties of the sample related to the refractive index thereof can be analyzed by finding the above-mentioned specific incidence angle θsp at which ATR takes place.
In the case of the leaky mode sensor, as with the case of the aforementioned surface plasmon resonance measuring apparatus, the cladding layer and the optical waveguide layer can be fixed to the dielectric block and formed into a single measuring unit. After a measurement of each sample is made, each measuring unit can be discarded.
In the field of pharmaceutical manufacture and the like, the above-mentioned surface plasmon resonance measuring apparatus and leaky mode sensor are sometimes used in a random screening for detecting a specific substance that bonds with a predetermined sensing substance. In this case, the sensing substance is placed on the aforementioned thin film layer (i.e., the metal film in the case of the surface plasmon resonance measuring apparatus, or the cladding layer and optical waveguide layer in the case of the leaky mode sensor). Then, a liquid sample containing a target substance is dropped into the sensing substance, and each time a predetermined time elapses, the aforementioned specific incidence angle θsp is measured.
If the target substance in the liquid sample bonds with the sensing substance, the refractive index of the sensing substance varies with the lapse of time by the bond therebetween. Therefore, every time a predetermined time elapses, the specific incidence angle θsp is measured. Based on the measured value, the bond between the target substance and the sensing substance is measured. Next, based on the result, it can be judged whether or not the target substance is a specific substance that bonds with the sensing substance. An example of combination of the specific substance and the sensing substance is an antigen and an antibody. As an example of a measurement of such combination, there is a measurement of the bond between a human IgG (immunoglobulin G) antibody in a target substance and a rabbit antihuman IgG antibody (sensing substance).
Note that the specific incidence angle θsp at which ATR occurs itself does not always need to be detected to measure the bond between the target substance and the sensing substance. For example, a liquid sample is added to the sensing substance; then a change in the specific incidence angle θsp thereafter is measured; and based on the angle change, the bond can be measured.
However, a measuring apparatus, such as the aforementioned surface plasmon resonance sensor and leaky mode sensor, has the disadvantage that when measuring a plurality of samples, the measurement is extremely time-consuming. Particularly, in the case in which a single sample is measured several times at predetermined temporal intervals in order to detect a change in the properties of the sample due to an antigen-antibody reaction, a chemical reaction, etc., a new sample cannot be measured unless the measurement of the single sample is finished, and consequently, it takes too much time to measure all samples.
In addition, in the case where a conventional measuring chip such as that mentioned above is employed, it is fairly difficult to position the measuring chip accurately in a measuring apparatus such as a surface plasmon resonance measuring apparatus, etc. In the measuring apparatus utilizing ATR, to make high-precise measurements with good reproducibility, there is a need to make a light beam strike the interface between the dielectric block and the thin film layer (which is the metal film in the case of a measuring apparatus utilizing surface plasmon resonance, or the cladding layer in the case of a measuring apparatus utilizing the excitation of a waveguide mode) in a predetermined incidence angle range. However, if it is difficult to position the measuring chip precisely in the measuring apparatus, the incidence angle range will vary, resulting in a reduction in accuracy of measurement. Furthermore, a conventional measuring chip such as that mentioned above is not convenient to handle. Because of this, it is difficult to make measurements efficiently.